REPRODUCTIVE HORMONES

Reproductive hormones fall into two broad categories based on their chemical
composition - protein and steroid; by definition, these compounds are secreted
and gain access to the circulation. Protein hormones are subclassified as neurohormones,
gonadotropins, or gonadal proteins. Neurohormones include GnRH, PRF, PIF,
oxytocin, and melatonin. Gonadotropic hormones can be of either pituitary (FSH,
LH, PRL) or placental (PMSG, hCG) derivation. In addition to steroid hormones,
the gonads produce inhibin, relaxin, and MIH. The chemical dispositions, sites
of synthesis and action, and most notable biological effects of the
reproductive hormones are summarized in Table 2-2. Figure 2-7 portrays a more comprehensive version of the
"hierarchy of control" of reproduction than presented earlier (Figure 1-2).

Protein synthesis and secretion. Synthesis of protein hormones (and
proteins in general) involves a series of complex steps whereby information
contained within the nucleotide makeup of DNA is expressed within a specified
sequence of building blocks, the amino acids (Table 2-3);
the product is assembled by two processes - transcription and translation.

Genes of eukaryotic (nucleated) cells contain both coding (exons, for EXtra
nuclear or EXpressed sequences) and intervening noncoding (introns) regions.
Before mRNA is exported from the nucleus, conveying a code for organization of
amino acids at the rough endoplasmic reticulum (translation), primary RNA
transcripts are cut and spliced into mature mRNAs (ie., introns are removed).

Triplet sequences of bases (codons) provide the genetic information encoded
into mRNA that is necessary for polymerization of specific amino acids into a
designated sequence. A given amino acid can have more than one codon. There are
61 codons for 20 amino acids in the general code. Ribosomes containing RNA
(rRNA) provide structural support for mRNA. Transfer RNAs (tRNA) containing
appropriate anticodons transport and attach amino acids to the elongating
protein. During translation the ribosome advances step-wise along the strand of
mRNA, a single codon (reading frame) at a time, beginning with the initiation
codon ATG (which specifies methionine). Synthesis of protein starts at the 5'
and terminates at the 3' end of mRNA. Nonsense codons (UAA, UGA, UAG), not
corresponding to an amino acid, serve to stop the transcriptional message. A
consensus sequence (AATAA) in the 3' untranslated region is the signal for
polyadenylation of mRNA. The addition of a poly-A tail is required for mRNA to
leave the nucleus (Figure 2-8).

Whereas the DNA content (blueprint) of somatic cells of an individual is identical
(excluding random mutations), genes along the helix that can be regulated vary
depending upon type of cell; this provides a basis for specialized function
among different cells and tissues. In other words, the primary sequences of
genes are the same from cell-to-cell - it is the ways they're read that differs. Transcription is suppressed when DNA is
bound by protein and rendered inaccessible by a polymerase ("the reader"). The majority of
sites along the genome (~ 95%) are masked by histones. Histone (basic) proteins
carry a strong positive charge, that attracts DNA. Structures of most nuclear
histones are well conserved within and between species. Enzymatic methylation
of cytosine bases of DNA (an inherited trait) is also important in the turning
on-or-off of specific genes. Inactive genes tend to be highly methylated. The
processes of how genes are activated (or suppressed) is called epigenetics.

Protein synthesized along the ribosome is transferred into and through the
endoplasmic reticulum and Golgi complex. Translated molecules are often
modified in transit through the endoplasmic reticulum and Golgi; this can
involve enzymatic modification (eg., cleavage of an active peptide hormone from
a larger prohormone), folding (eg., formation of covalent disulfide bonds
between residues of cysteine), derivatization (eg., addition of carbohydrate),
and(or) subunit assembly.

Protein hormones are packaged into secretory granules within the Golgi
apparatus. The cytoskeleton guides (microtubules) and provides a propulsive
force (microfilaments) for intracellular transport of granules (Figure 2-9). Secretory granules fuse with the cellular
membrane and release their hormonal contents into the extracellular space
(exocytosis). Residual hormone is stored in granules and thereby made readily
available for secretion upon a subsequent bout of cellular stimulation.
Receptors can be processed similarly and incorporated into the cellular
membrane (other receptors, enzymes, and structural elements are retained within
the cell).

Peptide bonds linking amino acids are susceptible to enzymatic attack.
Proteolysis is the major mechanism of inactivation of protein hormones; this
can occur within the circulation, liver, kidney, or target tissue. Terminal
residues of sialic acid on glycosylated proteins are removed by plasma
glycosidases. Hepatic cells recognize the exposed asialo hormone. Protein
hormones typically circulate in free form (ie., not bound to transport
molecules).

Neurohormones. Vertebrate GnRHs are composed of 10 amino acids. Only
one form of decapeptide has been described in eutherian mammals (Figure 2-10). In some species GnRH is found outside the
brain (eg., within the gonads [gonadocrinins] and placenta). An
extrahypothalamic version of GnRH, with sequence homology to chicken GnRH II,
has been reported in marsupials.

Gonadotropin-releasing hormone is cleaved from a prohormone during
posttranslational processing. The mature hormone acts on gonadotropes to induce
synthesis and secretion of FSH and LH. The first three amino acids of GnRH are
involved in activation of a response. Antagonistic analogs of GnRH (bind the
receptor, but do not elicit a response) are produced by substituting amino
acids in positions 1-3. Amino acids at positions 1 and 10 recognize binding
regions on the receptor. Affinity of GnRH for the receptor is amplified by
substitution of ethylamide at position 10. Peptide bonds associated with
glycine at position 6 are susceptible to enzymatic attack; replacement in this
position (eg., with D-Ala) will yield an analog that is resistant to enzymatic
hydrolysis (and has a better configuration for interaction with the receptor
than native GnRH). Synonyms of GnRH include LHRH, LHRH/FSHRH, gonadoliberin,
and luliberin.

There has been interest for some time in the concept that more than one
hypothalamic hormone controls FSH and LH. Putative releasing factors specific
for FSH, and an inhibitory hormone of LH, have not been purified to
homogeneity.

Production of prolactin is controlled by opposing hypothalamic forces. The
unequivocal chemical character of prolactin regulating factors has been the
subject of debate. Many studies indicate that PIF is dopamine (DA), while
others implicate g -aminobutyric acid
(GABA). A GnRH-associated peptide (GAP) that is contained within the sequence
of the GnRH prohormone also has PIF activity. Thyrotropin-releasing hormone
(TRH), vasointestinal peptide (VIP), and 5-hydroxytryptamine (serotonin) cause
release of prolactin. The PIF input is of greater physiological significance. A
recently discovered pituitary adenyl cyclase-activating polypeptide that
belongs to the VIP/secretin/glucagon family stimulates all anterior pituitary
cell types.

Oxytocin is composed of nine amino acids; it is assembled on the ribosomes
as part of a large precursor that includes its carrier protein, neurophysin.
The oxytocin-neurophysin complex is packaged into neurosecretory granules.
Neurophysin is cleaved from oxytocin after axonal transport to the
neurohypophysis. A disulfide bond links the cysteine residues of oxytocin at
positions 1 and 6 (Figure 2-11) - giving the molecule
a ring structure that is required for biological activity. Oxytocin stimulates
contraction of uterine smooth muscle aiding in expulsion of the fetus during
labor and stimulates milk ejection by contracting myoepithelial cells that
surround mammary alveoli. Obligatory effects of oxytocin in the male have not
been demonstrated. Oxytocin has been isolated within the periphery (gonads,
placenta, adrenal gland, uterus, male accessory glands). The plasma half-life
of oxytocin is < 5 minutes.

In contrast to the hypothalamic neurohormones described above, melatonin is
a product of the pineal gland. The pineal gland is located within the roof of
the third ventricle (Figure 2-2). Like the OVLT, the
pineal gland is a circumventricular organ, and lies outside the blood-brain
barrier (the majority of endothelial cells lining CNS capillaries are sealed by
tight junctions, restricting entry of large molecules). Melatonin, an
indolamine, is synthesized by enzymatic processing of tryptophan. Tryptophan is
converted into serotonin. Serotonin is modified into melatonin by the dual
action of two enzymes - N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase
(HIOMT) (Figure 2-12). Monoamines are inactivated by
enzymatic methylation and oxidation. Melatonin is hydroxylated and further
conjugated with sulfate. Melatonin participates in the control of seasonal
reproduction in some species.

Gonadotropins. Gonadotropins (and TSH) of advanced vertebrates are
composed of an a and b subunit. Amino acid sequences of a subunits are well conserved (ie., have
evolved from a common ancestral gene). It is the b
moiety of gonadotropins that imparts specificity to the molecule (interchange
of a subunits with a b yields activity intrinsic to b). Synthesis of mammalian a and b
subunits occurs from mRNAs transcribed from genes of different chromosomes.
Subunits are glycosylated, compacted by intrachain disulfide bonds, and
assembled noncovalently. Synthesis of b
subunit is limiting to overall production of hormone. Free b subunit will bind (with reduced affinity)
to the gonadotropin receptor; however, glycosylated subunits must be joined to
elicit a biological response.

The half-life of the placental gonadotropins within the circulation is
longer than for their counterpart pituitary hormones (eg., FSH @ 45 minutes, PMSG = 1-6 days). Additional
sialic acid residues shield the placental molecules from metabolic clearance.
The placental gonadotropins also have an extended C-terminus (eg., masses of LH
and hCG are about 28 and 37 kilodaltons [kDa], respectively).

Human choriogonadotropin (Figure 2-13) binds to
luteal LH receptors and stimulates progesterone production - sustaining early
pregnancy. Analogous chorionic hormones have been isolated in other primates
and the guinea pig. The function of equine choriogonadotropins (horse, donkey,
zebra) in pregnancy is indefinite. Horse CG is thought to cause follicular
growth and luteinization (ie., formation of accessory or secondary CL) during
early gestation (PMSG has both FSH- and LH-like activities).

Prolactin is a single-chain protein that is folded by three disulfide bonds
(Figure 2-14); it does not contain residues of
carbohydrate. Prolactin is similar in structure to GH and placental lactogens.
Prolactin is classified as a gonadotropin in that it stimulates steroidogenic
function of the CL in certain species (eg., rodents). Milk synthesis and
induction of maternal behavior in farm animals are induced by prolactin.
Hyperprolactinemia in humans is associated with infertility, galactorrhea
(production of milk outside the normal postpartum period), and gynecomastia
(excessive breast development in males); the condition is treated with an ergot
alkaloid, cabergoline or bromocryptine (CB-154), dopamine agonists.

Gonadal proteins. Inhibin is a glycoprotein with a molecular mass of
approximately 32 kDa composed of two subunits coupled by disulfide bridges. The
primary structure of inhibin has been characterized for some species (murine,
porcine, bovine, human; Figure 2-15). Inhibin is produced
by the testis and ovary. Inhibin of ovarian origin is sometimes called
folliculostatin. Two forms of inhibin (A and B) have been isolated.
Glycosylated a subunits of each type of
inhibin are identical. However, two homologous, but different, unglycosylated
inhibin b subunits have been described.
Sequences of inhibin subunits are highly conserved between species. Subunits
for inhibin are encoded by different mRNAs (probably descendent from one gene).
Inhibin is derived from larger precursor molecules. Inhibin is a chalone - a
hormone that inhibits rather than stimulates; it specifically suppresses
secretion of FSH by acting at the level of the anterior pituitary gland.
Diverse patterns of secretion of FSH and LH result from differential control of
the gonadotrope imposed by inhibin.

Gonadostatin is a gonadal factor reported to inhibit secretion of both FSH
and LH. Several other variants of inhibin have been identified. Dimers formed
between inhibin b subunits (activins)
exert FSH-releasing properties. Beta subunits of inhibin are structurally
similar to transforming growth factor (TGF) b
and MIH.

Relaxin is a polypeptide hormone (~
6000 daltons) produced by the ovaries and placenta; it is made up of two
dissimilar subunits (A and B) connected by a pair of disulfide bonds. Three
forms of porcine relaxin have been isolated that differ slightly in length of
the C terminus of the B chain. Relaxin is synthesized as a preprohormone (Figure 2-16). There is structural homology between
relaxin, insulin, and the somatomedins. Structure and potency of relaxin is
poorly conserved across species. Relaxin causes dilation of the cervix,
relaxation of pelvic ligaments, and coordinates uterine contractions during the
birth process.

Mullerian-inhibiting hormone is a disulfide-linked homodimeric glycoprotein
of 140 kDa synthesized by the fetal testis. A prepro leader sequence and
bioactive C-terminal fragments are cleaved from the mature MIH protein.
Regression of the female duct system of the sexually-indifferent male embryo is
induced by MIH.

Steroidogenesis. Steroid hormones are derived from cholesterol.
Circulatory cholesterol (of dietary origin or synthesized from acetate within
the liver) is bound as esters within low density lipoprotein (LDL).
Cholesterol-LDL complexes are internalized into the cell by receptor-mediated
endocytosis. Receptors oriented within the cellular membrane bind extracellular
ligand, move by lateral diffusion, aggregate within discrete zones (such areas
are sometimes associated with a coating of clathrin along the inner aspect of
the cellular membrane - "coated pits"), and form vesicles that
eventually pinch off toward the interior of the cell. Endocytotic vesicles lose
their coating and coalesce to form an endosome. The endosome then fuses with
primary lysosomes (specialized vesicles of the Golgi complex that contain acid
hydrolases) to make a secondary lysosome. Ligand-receptor complexes are
dissociated within secondary lysosomes and cholesterol esters are enzymatically
cleaved from LDL. Receptors are retained within the vesicle and recycled back
to the cellular membrane (Figure 2-17). Fat droplets
(Figure 2-18) serve as an intracellular reservoir of
cholesterol esters. Only free (hydrolyzed) cholesterol can be used as substrate
for biosynthesis of steroid hormones. Cholesterol is transferred to the inner
mitochondrial membrane by steroidogenic acute regulatory protein (StAR).

Pregnenolone is derived in mitochondria by enzymatic modification of
cholesterol; it is then translocated to the smooth endoplasmic reticulum for
further processing into progestogens, androgens, or estrogens. Thus,
pregnenolone is a common precursor for gonadal steroidogenesis (Figure 2-19). Primary bioactive secretory products of
the CL, testis, and follicle are progesterone (4-pregnen-3, 20-dione),
testosterone (17b-hydroxy-4-androsten-3-one),
and estradiol (1, 3, 5[10]-estratrien-3, 17b-diol),
respectively. The placenta is a major source of steroid hormones during
pregnancy.

It has been assumed that steroid hormones are secreted from the cell by
passive diffusion (ie., down a concentration gradient). However, there is some
evidence that steroids are packaged into secretory granules and released from
the cell by exocytosis.

Steroid hormones are metabolized primarily within the liver. The steroid
molecule is inactivated by saturation. Conjugation with sulfates or
glucuronates makes the steroid hormone water soluble, so it can be excreted
(high density lipoprotein apparently targets cholesterol for hepatic
metabolism). In some cases circulatory steroid hormones are converted to a more
active compound within a target tissue (Figure 2-20).

Gonadal steroid hormones carry out an array of activities - primary effects
are on maintenance of reproductive tracts, feedback regulation of secretion of
gonadotropins, and behavior. Secondary sex characteristics are also altered by
steroids. Female sex steroids interact with other hormones to stimulate growth
and development of mammary tissues.

Cortisol is not usually classified as a reproductive hormone; its main
actions are in organic metabolism and antiinflammatory/immune responses. Notwithstanding,
cortisol originating from the fetal adrenal cortex has been implicated in the
triggering mechanism of labor in some farm species. Adrenal 21- and 11b-hydroxylases convert 17a-hydroxyprogesterone to cortisol.